Modified membrane spanning proteins and methods for the preparation and use thereof

ABSTRACT

In accordance with the present invention, there are provided functionally modulated tool receptors which are useful for drug discovery and development. In certain aspects and embodiments as described herein, a sophisticated and powerful approach has been designed that allows the rapid development of enhanced receptors, while simultaneously exploring millions of possibilities for improved properties with respect to such properties as protein expression, homogeneity, stabilization, conformational and activation pathway selectivity, antigenicity, immunogenicity, and the like. Indeed, the new methodology described herein represents a breakthrough by leveraging a full range of combinatorial amino acid replacements, in multiple positions simultaneously, in order to generate modified membrane-spanning proteins.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/411,059, filed May 13, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/928,128, (now U.S. Pat. No. 10,287,349) filedOct. 30, 2015, which claims the benefit of U.S. Provisional ApplicationNo. 62/073,554, filed Oct. 31, 2014, each of which application isincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to methods to modulate the functionalproperties of membrane-spanning proteins. In one aspect, the presentdisclosure relates to modified membrane-spanning proteins. In anotheraspect, the present disclosure relates to methods for the use of suchmodified membrane-spanning proteins.

BACKGROUND OF THE INVENTION

The information provided herein and references cited are provided solelyto assist the understanding of the reader, and does not constitute anadmission that any of the references or information is prior art to thepresent invention.

Membrane-spanning proteins such as G Protein Coupled Receptors (GPCRs)are among the most important classes of drug targets, currently targetedby about 30% of therapeutics in the market. However, the use of GPCRsfor basic drug discovery and development in the pharmaceutical industryis inherently difficult due to the notoriously low expression,homogeneity, and stability thereof, and the high conformationalflexibility of these integral membrane proteins, especially when removedfrom the cellular environment. This has greatly limited the success ofprotein-based approaches such as small molecule screening/assays andstructural determination for structure-guided drug discovery, as well asantibody discovery efforts. These limitations remain to be addressed.

Importantly, only a very limited number of therapeutic antibodiestargeting GPCRs have been approved, while most marketed therapeutics aresmall molecules or small peptides. As experienced often with non-GPCRtargets, biologic drugs such as antibodies may enable access to atherapeutic space where small molecule drugs have failed. Therefore, theavailability of GPCR-targeting antibodies will potentially facilitatepreviously unattainable opportunities for the betterment of humanhealth.

SUMMARY OF THE INVENTION

In accordance with the present invention, the limitations of the arthave been addressed through the generation of stabilized tool receptorsfor use in drug discovery and development.

Therefore, in certain aspects and embodiments as described herein, asophisticated and powerful approach has been designed that allows therapid development of enhanced receptors, while simultaneously exploringmillions of possibilities for improved properties, such as increasedprotein expression, increased protein stability, increased proteinhomogeneity, increased protein antigenicity, increased proteinimmunogenicity, increased protein crystallizability, modulatedconformational selectivity, modulated activation pathway selectivity,and the like. Indeed, the new methodology described herein represents abreakthrough by leveraging a full range of combinatorial amino acidreplacements, in multiple positions simultaneously, in order to generatemodified membrane-spanning proteins.

In addition, the efficiency of the readout system employed herein, i.e.,the system by which clones are identified, is exponentially improvedover current screening methods. Therefore, besides achieving higherprotein expression and trafficking, the resulting membrane-spanningproteins (e.g., GPCRs) are characterized by having a greater spectrum ofvariation with respect to properties such as, for example, higherstability, a higher degree of conformational selectivity, and a higherdegree of homogeneity), and the like. Therefore, modifiedmembrane-spanning proteins, as described herein have great potential toaid the development of both biologics, small peptides and small moleculedrugs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a GPCR family tree adopted from ihuman institute of ShanghaiTech.

FIG. 2 presents a schematic diagram of two exemplary constructs preparedas described in Example 2, for the expression of membrane proteins in abacterial selection host, in their wild type or enhanced mutated form.START and STOP refer to translation initiation and termination; boxesindicate genes which translate into functionally independent proteins.

FIG. 3 presents a Western blot of a wild type receptor and its enhancedmutant (also referred to herein as “Enabled Membrane Protein” or “EMP”).Staining of the FLAG-tagged fusion EMP was carried out using ananti-FLAG HRP-conjugated antibody. The protein marker indicatesmolecular weights in KDa.

FIG. 4 presents thermal unfolding curves measured using fluorescent sizeexclusion chromatography (fSEC) on purified receptors in aprotein-detergent complex after one round of mutagenesis. The wild typereceptor is shown in closed circles, whereas a stabilized mutant isshown in triangles.

FIG. 5 presents normalized fSEC peaks of purified wild type (blacktrace) and mutant receptors (EMPs). The peaks corresponding to monomerand dimer of the analyzed GPCR (estimated oligomeric state of theanalyzed samples) are indicated with a black arrow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there are provided methods tomodulate the functional properties of a membrane-spanning protein, saidmethods comprising:

-   -   generating a first polynucleotide library wherein a sufficient        number of residues of a polynucleotide encoding said        membrane-spanning protein are randomly modified so as to        modulate the functional properties thereof,    -   optionally generating a second polynucleotide library from said        first polynucleotide library, said second polynucleotide library        comprising a polynucleotide encoding said membrane-spanning        protein modified by DNA shuffling in a sufficient number of        amino acid residues so as to modulate the functional properties        thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence,        -   a first marker sequence, wherein said first marker sequence            is in-frame with said modified polynucleotide, and encodes            one or more genes that overcome the sensitivity of said host            to the presence of selective pressure agent(s)/conditions,            and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions and identifying the modified        polynucleotide-containing construct(s) contained therein.

As used herein, the phrase “membrane-spanning protein” refers to plasmamembrane proteins, nuclear membrane proteins, peripheral membraneproteins, intracellular-membrane proteins (e.g. mitochondrial membraneproteins), transporters, channels, adhesins, translocases and receptorslike G Protein Coupled Receptors (GPCRs) from all classes (A-F).Exemplary G-protein coupled receptors (GPCR) include adhesion receptors,secretins, glutamates, frizzled/TAS2, rhodopsin, olfactory receptors,and the like. See, for example, the GPCR family tree set forth in FIG.1.

In generating a first polynucleotide library wherein a sufficient numberof residues of a polynucleotide encoding said membrane-spanning protein(and the corresponding polypeptide sequence after translation thereof)are randomly modified so as to modulate the functional propertiesthereof, one typically modifies in the range of 1-50 residues; in someembodiments, one typically modifies 1-40 residues; in some embodiments,one typically modifies 1-30 residues; in some embodiments, one typicallymodifies 1-20 residues; in some embodiments, one typically modifies 1-15residues; in some embodiments, one typically modifies 2-50 residues; insome embodiments, one typically modifies 2-40 residues; in someembodiments, one typically modifies 2-30 residues; in some embodiments,one typically modifies 2-20 residues; in some embodiments, one typicallymodifies 2-15 residues; in some embodiments, one typically modifies 3-50residues; in some embodiments, one typically modifies 3-40 residues; insome embodiments, one typically modifies 3-30 residues; in someembodiments, one typically modifies 3-20 residues; in some embodiments,one typically modifies 3-15 residues; in some embodiments, one typicallymodifies 4-50 residues; in some embodiments, one typically modifies 4-40residues; in some embodiments, one typically modifies 4-30 residues; insome embodiments, one typically modifies 4-20 residues; in someembodiments, one typically modifies 4-15 residues; in some embodiments,one typically modifies 5-50 residues; in some embodiments, one typicallymodifies 5-40 residues; in some embodiments, one typically modifies 5-30residues; in some embodiments, one typically modifies 5-20 residues; insome embodiments, one typically modifies 5-15 residues; an average of 6residues are modified (mutated) per 1 kB of DNA; in some embodiments, anaverage of 8 residues are modified (mutated) per 1 kB of DNA; in someembodiments, an average of 10 residues are modified (mutated) per 1 kBof DNA; in some embodiments, an average of 12 residues are modified(mutated) per 1 kB of DNA.

In optionally generating a second polynucleotide library from said firstpolynucleotide library, said second polynucleotide library comprising apolynucleotide encoding said membrane-spanning protein modified by DNAshuffling in a sufficient number of amino acid residues so as tomodulate the functional properties thereof, one typically modifies 1-50residues; in some embodiments, one typically modifies 1-40 residues; insome embodiments, one typically modifies 1-30 residues; in someembodiments, one typically modifies 1-20 residues; in some embodiments,one typically modifies 1-15 different amino acid residues perpolynucleotide. In some embodiments, one typically modifies 2-50residues; in some embodiments, one typically modifies 2-40 residues; insome embodiments, one typically modifies 2-30 residues; in someembodiments, one typically modifies 2-20 residues; in some embodiments,one typically modifies 2-15 different amino acid residues perpolynucleotide; in some embodiments, one typically modifies 3-50residues; in some embodiments, one typically modifies 3-40 residues; insome embodiments, one typically modifies 3-30 residues; in someembodiments, one typically modifies 3-20 residues; in some embodiments,one typically modifies 3-15 different amino acid residues perpolynucleotide; in some embodiments, one typically modifies 4-50residues; in some embodiments, one typically modifies 4-40 residues; insome embodiments, one typically modifies 4-30 residues; in someembodiments, one typically modifies 4-20 residues; in some embodiments,one typically modifies 4-15 different amino acid residues perpolynucleotide; in some embodiments, one typically modifies 5-50residues; in some embodiments, one typically modifies 5-40 residues; insome embodiments, one typically modifies 5-30 residues; in someembodiments, one typically modifies 5-20 residues; in some embodiments,one typically modifies 5-15 different amino acid residues perpolynucleotide.

As used herein, “signal sequence” refers to a sequence of amino acidresidues in the amino terminus of a nascent protein during proteintranslation, which when recognized by the signal recognition particleresults in the transport of the nascent protein via the translocationpathway of the host organism.

Marker sequences contemplated for use herein encode one or more genesthat overcome the sensitivity of said host to the presence of selectivepressure agent(s) and/or selective pressure conditions. Exemplary markersequences include ampicillin, chloramphenicol, neomycin, kanamycin,tetracycline, gentamicin resistance genes; pyrE gene (orotatephosphoribosyltransferase) and pyrF gene (orotidine-5′-monophosphatedecarboxylase); URA3 gene (orotidine 5′-phosphate decarboxylase); LYS2gene (alpha-aminoadipate reductase); ADE1-2 genes(phosphoribosylamino-imidazole-succinocarbozamide synthetase,phosphoribosylamino-imidazole-carboxylase), and the like.

Selective pressure agents/conditions refer to agents (includingconditions) of differential mortality or fertility that tend to make apopulation change genetically. Exemplary selective pressure agentsinclude antibiotics (e.g., ampicillin, carbenicillin, gentamicin,chloramphenicol, neomycin, kanamycin, tetracycline, and the like), toxicmetabolites (e.g., 5-fluoroorotic acid or uracyl), conditions such aslack of nutrients for auxotrophic strains, and the like.

Exemplary selective pressure conditions include elevated temperature,reduced temperature, lack of necessary nutrient(s), co-factors, and thelike. Exemplary nutrients include oxygen, carbon dioxide, and the like.

Other marker sequences contemplated for use herein encode reporter genesthat confer traits that can be easily identified and measured inorganisms expressing same. Exemplary marker sequences includebeta-galactosidase, alkaline phosphatase, green fluorescent protein, redfluorescent protein, tdTomato fluorescent protein, luciferase, and thelike.

Inserting genetic material into suitable host cells with the modifiedpolynucleotide-containing constructs described herein can be carried outin a variety of ways, e.g., by transformation or transfection, theprocess by which nucleic acids are introduced into bacteria or mammaliancells, respectively. Protocols and techniques vary widely and includelipid transfection and chemical and physical methods such aselectroporation.

A variety of techniques suitable to employ for selecting cells whichsurvive exposure to the selective pressure agent(s)/conditions are wellknown in the art (e.g. cell growth on agar plates), as are methods foridentifying the modified polynucleotide-containing construct(s)contained therein according to the present invention (e.g. the Sangerpolynucleotide sequencing method).

In some embodiments of the present invention, the functionalproperty(ies) of a membrane-spanning protein are selected from theexpression level of said membrane-spanning protein, the stability ofsaid membrane-spanning protein, the conformational selectivity of saidmembrane-spanning protein, the homogeneity of said membrane-spanningprotein, the crystallizability of said membrane-spanning protein, theantigenicity of said membrane-spanning protein, the immunogenicity ofsaid membrane-spanning protein, the activation pathway selectivity ofsaid membrane-spanning protein, and the like.

In some embodiments of the present invention, additional assays arecarried out to verify the occurrence of membrane-spanning proteinshaving modulated functional properties. Such verification can be carriedout, for example, by:

-   -   inserting the modified polynucleotide into suitable host cells,    -   expressing said modified polynucleotide, and    -   characterizing the resulting protein.

As readily recognized by those of skill in the art, a variety ofcharacterization techniques and protocols are available to assess thevarious properties of the membrane proteins developed employinginvention methods, e.g., homogeneity estimation and thermal denaturationassays, analytical size exclusion chromatography (SEC) or fluorescentSEC or fluorescent dyes; gel electrophoresis; direct bindingmeasurements (including radioligand binding assays and surface plasmonresonance); signal transduction measurements (including cAMP productionand calcium flux assays); and the like.

In some embodiments of the present invention, additional assays arecarried out to identify the minimum working set of mutations required toachieve the desired modulated functional properties. Such evaluation canbe carried out, for example, by:

-   -   randomly reverting said modified residues back,    -   re-selecting in the presence of selective pressure        agent(s)/conditions (e.g., antibiotic-containing medium), and    -   re-characterizing (the modulated functional properties) in order        to identify the minimum working set of mutations.

As readily recognized by those of skill in the art, a variety of hostcells are suitable for use in the invention methods. Exemplary hostcells are typically characterized by one or more of the followingcriteria:

-   -   being able to undergo transfection, transformation etc to        receive (exogenous) genetic material (DNA, mRNA, plasmids,        bacmids etc.);    -   being able to receive a limited number of copies of genetic        material;    -   being able to form colonies or clonal biomass (genetically        uniform);    -   possessing one or more selection markers (e.g., antibiotics,        auxotrophies, toxins);    -   being able to effectively produce transmembrane proteins;    -   possessing the appropriate cellular machinery for translocation        of proteins to the membrane environment; and/or    -   being able to grow in mesophilic, thermophilic,        hyperthermophilic and/or other extreme conditions.

Suitable host cells contemplated for use herein can be selected from thegroup consisting of bacterial cell lines, yeast cell lines, insect celllines, mammalian cell lines, and the like.

Exemplary bacterial cell lines include, for example, Escherichia coli,Bacillus subtilis, Baccillus brevis, Bacillus megaterium, Pseudomonasfluorescens, Thermus thermophilus, Aeropyrum pernix, Corynebacteriumglutamicum, Sulfolobus islandicus, and the like.

Exemplary yeast cell lines include, for example, Pichia methanolica,Pichia angusta, Pichia thermomethanolica, Pichia pastoris, Saccharomycescerevisiae, Schizosacchamromyces pombe, Arxula adeninivorans,Kluyveromyces lactis, Yarrowia hpolytica, and the like.

Exemplary mammalian cell lines include, for example, Embryonic Kidneycells (Human), Bos primigenius (Bovine), Mus musculus (Mouse),Cricetulus griseus (Chinese Hamster Ovary), Baby Hamster Kidney, and thelike.

Exemplary insect cells include, for example, Spodoptera frupperda,Trichoplusia ni, and the like.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the functional properties of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        modulate the functional properties thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions,        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the functional properties of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        modulate the functional properties thereof, said construct        comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the functional properties of amembrane-spanning protein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequences,    -   wherein:        -   said marker sequence is in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to modulate the functional properties thereof, and            optionally thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to modulate the functional        properties thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the functional properties of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditons (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequences,

-   -   wherein:        -   said marker sequence is in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to modulate the functional properties thereof, and            optionally thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to modulate the functional        properties thereof.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein.

In certain aspects of the present invention , wherein the property to bemodulated is the stability of said membrane-spanning protein, theinvention method comprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to modulate the stability thereof,    -   optionally generating a second polynucleotide library from said        first polynucleotide library, said second polynucleotide library        comprising a polynucleotide encoding said membrane-spanning        protein modified by DNA shuffling in a sufficient number of        amino acid residues so as to modulate the stability thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence,        -   a first marker sequence, wherein said first marker sequence            is in-frame with said modified polynucleotide, and encodes            one or more genes that overcome the sensitivity of said host            to the presence of selective pressure agent(s)/conditions,            and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased stability relativeto wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the stability of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        modulate the stability thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased stability relativeto wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the stability of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        modulate the stability thereof, said construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased stability relativeto wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the stability of a membrane-spanningprotein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequences,    -   wherein:        -   said marker sequence is in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the stability thereof, and optionally thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the stability        thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased stability relativeto wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the stability of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditions (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequences,

-   -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein are randomly modified so as to modulate the            stability of said membrane-spanning protein, and thereafter            optionally    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the stability        thereof.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased stability relativeto wild type.

In certain aspects of the present invention, wherein the property to bemodulated is the homogeneity of a membrane-spanning protein, theinvention method comprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to increase the homogeneity thereof,    -   optionally generating a second polynucleotide library from said        first polynucleotide library, said second polynucleotide library        comprising a polynucleotide encoding said membrane-spanning        protein modified by DNA shuffling in a sufficient number of        amino acid residues so as to increase the homogeneity thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence,        -   a first marker sequence, wherein said first marker sequence            is in-frame with said modified polynucleotide, and encodes            one or more genes that overcome the sensitivity of said host            to the presence of selective pressure agent(s)/conditions,            and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions, (e.g., an antibiotic) and        identifying the modified polynucleotide-containing construct(s)        contained therein.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningproteins have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased homogeneityrelative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the homogeneity of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        increase the homogeneity thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased homogeneityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the homogeneity of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        increase the homogeneity thereof, said construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent/condition (e.g., antibiotic) and identifying the        modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased homogeneityrelative to wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the homogeneity of a membrane-spanningprotein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequences,    -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the homogeneity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the homogeneity        thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased homogeneityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the homogeneity of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditions (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequence,

-   -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the homogeneity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the homogeneity        thereof.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased homogeneityrelative to wild type.

In certain aspects of the present invention, wherein the property to bemodulated is the crystallizability of a membrane-spanning protein, theinvention method comprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to increase the crystallizability thereof,    -   optionally generating a second polynucleotide library from said        first polynucleotide library, said second polynucleotide library        comprising a polynucleotide encoding said membrane-spanning        protein modified by DNA shuffling in a sufficient number of        amino acid residues so as to increase the crystallizability        thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence,        -   a first marker sequence, wherein said first marker sequence            is in-frame with said modified polynucleotide, and encodes            one or more genes that overcome the sensitivity of said host            to the presence of selective pressure agent(s)/conditions,            and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions, (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., higher crystallizabilityrelative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the crystallizability of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        increase the crystallizability thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., higher crystallizabilityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the crystallizability of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        increase the crystallizability thereof, said construct        comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., higher crystallizabilityrelative to wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the crystallizability of amembrane-spanning protein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequence(s),    -   wherein:        -   said marker sequence is in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the crystallizability thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the        crystallizability thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., higher crystallizabilityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the crystallizability of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditions (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequence(s),

-   -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the crystallizability thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the        crystallizability thereof

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., higher crystallizabilityrelative to wild type.

In certain aspects of the present invention, wherein the property to bemodulated is the antigenicity of a membrane-spanning protein, theinvention method comprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to increase the antigenicity thereof,    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein modified by DNA shuffling in a        sufficient number of amino acid residues so as to increase the        antigenicity thereof,    -   inserting the resultant modified genes into a construct        comprising:        -   a signal sequence,            -   a first marker sequence, wherein said first marker                sequence is in-frame with said modified polynucleotide,                and encodes one or more genes that overcome the                sensitivity of said host to the presence of selective                pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,    -   transforming suitable host cells with the resulting constructs,        and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased antigenicityrelative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the antigenicity of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        increase the antigenicity thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased antigenicityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the antigenicity of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        increase the antigenicity thereof, said construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased antigenicityrelative to wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the antigenicity of a membrane-spanningprotein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequence(s),    -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the antigenicity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the antigenicity        thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased antigenicityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the antigenicity of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditions (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequence(s),

-   -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the antigenicity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the antigenicity        thereof

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased antigenicityrelative to wild type.

In certain aspects of the present invention, wherein the property to bemodulated is the immunogenicity of a membrane-spanning protein, theinvention method comprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to increase the immunogenicity thereof,    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein modified by DNA shuffling in a        sufficient number of amino acid residues so as to increase the        immunogenicity thereof,    -   inserting the resultant modified genes into a construct        comprising:        -   a signal sequence,            -   a first marker sequence, wherein said first marker                sequence is in-frame with said modified polynucleotide,                and encodes one or more genes that overcome the                sensitivity of said host to the presence of selective                pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,    -   transforming suitable host cells with the resulting constructs,        and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased immunogenicityrelative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the immunogenicity of amembrane-spanning protein, said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        increase the immunogenicity thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased immunogenicityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the immunogenicity of amembrane-spanning protein, said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        increase the immunogenicity thereof, said construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased immunogenicityrelative to wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the immunogenicity of amembrane-spanning protein, said method comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequence(s),    -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the immunogenicity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the        immunogenicity thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased immunogenicityrelative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the immunogenicity of amembrane-spanning protein, said methods comprising selecting those cellswhich survive exposure to selective pressure agent(s)/conditions (e.g.,antibiotic) and identifying the modified polynucleotide-containingconstruct(s) contained therein, wherein said cells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequence(s),

-   -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to increase the immunogenicity thereof, and optionally            thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to increase the        immunogenicity thereof.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., increased immunogenicityrelative to wild type.

In certain aspects of the present invention, wherein the property to bemodulated is the conformational selectivity and/or activation pathwayselectivity of a membrane-spanning protein, the invention methodcomprises:

-   -   generating a first polynucleotide library wherein a sufficient        number of bases of a polynucleotide encoding said        membrane-spanning protein and/or a sufficient number of amino        acid residues of said membrane-spanning protein are randomly        modified so as to modulate the conformational selectivity and/or        activation pathway selectivity thereof,    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein modified by DNA shuffling in a        sufficient number of amino acid residues so as to modulate the        conformational selectivity and/or activation pathway selectivity        thereof,    -   inserting the resultant modified genes into a construct        comprising:        -   a signal sequence,            -   a first marker sequence, wherein said first marker                sequence is in-frame with said modified polynucleotide,                and encodes one or more genes that overcome the                sensitivity of said host to the presence of selective                pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,    -   transforming suitable host cells with the resulting constructs,        and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

As used herein, “conformational selectivity” refers to the activityprofile of a polypeptide as a function of the conformation thereof. Forexample, certain conformations of a peptide or protein may be more orless susceptible to binding or activating other proteins, peptides orcompounds. In certain embodiments and aspects, the conformationalselectivity embraces samples for which the conformation thereof issubstantially homogeneous.

As used herein, “activation pathway selectivity” refers to the abilityof an agent to selectively activate one or a limited number of metabolicor signaling pathways from among a plurality of available metabolic orsignaling pathways.

Suitable host cells contemplated for use in this aspect of the presentinvention can be selected from the group consisting of bacterial celllines, yeast cell lines, insect cell lines, and mammalian cell lines.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., modulated conformationalselectivity and/or activation pathway selectivity relative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods to modulate the conformational selectivityand/or activation pathway selectivity of a membrane-spanning protein,said method comprising:

-   -   generating a polynucleotide library wherein a sufficient number        of bases of a polynucleotide encoding said membrane-spanning        protein and/or a sufficient number of amino acid residues of        said membrane-spanning protein are randomly modified so as to        modulate the conformational selectivity and/or activation        pathway selectivity thereof,    -   inserting the modified polynucleotides of said second        polynucleotide library into a construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., modulated conformationalselectivity and/or activation pathway selectivity relative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the conformational selectivityand/or activation pathway selectivity of a membrane-spanning protein,said methods comprising:

-   -   inserting each member of a modified polynucleotide library into        a construct, wherein a sufficient number of bases of a        polynucleotide encoding said membrane-spanning protein and/or a        sufficient number of amino acid residues of said        membrane-spanning protein are randomly modified so as to        modulate the conformational selectivity and/or activation        pathway selectivity thereof, said construct comprising:        -   a signal sequence, and        -   a first marker sequence, wherein said marker sequence is            in-frame with said modified polynucleotide, and encodes one            or more genes that overcome the sensitivity of said host to            the presence of selective pressure agent(s)/conditions, and        -   optionally a second marker sequence, wherein said second            marker sequence, when present, is downstream of said signal            sequence, but upstream of said modified polynucleotide,        -   thereby producing modified polynucleotide-containing            constructs,    -   transforming suitable host cells with the modified        polynucleotide-containing constructs, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., modulated conformationalselectivity and/or activation pathway selectivity relative to wild type.

In accordance with a further embodiment of the present invention, thereare provided methods to modulate the conformational selectivity and/oractivation pathway selectivity of a membrane-spanning protein, saidmethod comprising:

-   -   transforming suitable host cells with a construct comprising:        -   a modified polynucleotide,        -   a signal sequence, and        -   one or more marker sequence(s),    -   wherein:        -   said marker sequence(s) is/are in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to modulate the conformational selectivity and/or activation            pathway selectivity thereof, and optionally thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to modulate the        conformational selectivity and/or activation pathway selectivity        thereof, and    -   selecting those cells which survive exposure to said selective        pressure agent(s)/conditions (e.g., antibiotic) and identifying        the modified polynucleotide-containing construct(s) contained        therein.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., modulated conformationalselectivity and/or activation pathway selectivity relative to wild type.

In accordance with yet another embodiment of the present invention,there are provided methods to modulate the conformational selectivityand/or activation pathway selectivity of a membrane-spanning protein,said methods comprising selecting those cells which survive exposure toselective pressure agent(s) (e.g., antibiotic) and/or selective pressureconditions, and thereafter identifying the modifiedpolynucleotide-containing construct(s) contained therein, wherein saidcells are transformed with:

a modified polynucleotide,

a signal sequence, and

one or more marker sequence(s),

-   -   wherein:        -   said marker sequence is in-frame with said modified            polynucleotide, and encodes one or more genes that overcome            the sensitivity of said host to the presence of selective            pressure agent(s)/conditions,        -   said modified polynucleotide is obtained by generating a            first polynucleotide library wherein a sufficient number of            bases of a polynucleotide encoding said membrane-spanning            protein and/or a sufficient number of amino acid residues of            said membrane-spanning protein are randomly modified so as            to modulate the conformational selectivity and/or activation            pathway selectivity thereof, and optionally thereafter    -   generating a second library from said first library, said second        library comprising a polynucleotide encoding said        membrane-spanning protein randomly modified in a sufficient        number of amino acid residues so as to modulate the        conformational selectivity and/or activation pathway selectivity        thereof.

In accordance with yet another embodiment of the present invention,there are provided modified membrane-spanning proteins identified by theabove-described methods. Typically, said modified membrane-spanningprotein have one or more improved functional properties relative to thewild type membrane-spanning protein, e.g., modulated conformationalselectivity and/or activation pathway selectivity relative to wild type.

In accordance with still another embodiment of the present invention,there are provided methods of generating diagnostic and/or therapeuticantibodies, said methods comprising generating an antibody against anyof the modified membrane-spanning proteins described herein. Antibodygeneration can be carried out using techniques that are well known inthe art. Antibody selection can be greatly improved by usingthermostabilized mutants prepared employing invention methods. See, forexample, Hutchings C J et al. 2014 Mabs, 6(1):246-61).

In accordance with yet another embodiment of the present invention,there are provided methods of treating a disease or condition mediatedby a membrane-spanning protein, said method comprising administering aneffective amount of a therapeutic antibody prepared as described hereinto a subject in need thereof.

In accordance with yet another embodiment of the present invention,there are provided methods of treating a disease or condition mediatedby a membrane-spanning protein, said method comprising administering aneffective amount of a modified membrane-spanning protein as describedherein to a subject in need thereof.

In accordance with yet another embodiment of the present invention,there are provided methods of treating a disease or condition mediatedby a membrane-spanning protein, said method comprising administeringpolynucleotide encoding a membrane-spanning protein according to any ofthe modified membrane-spanning proteins described herein.

The following examples are provided to further illustrate aspects of theinvention. These examples are non-limiting and should not be construedas limiting any aspect of the invention.

EXAMPLE 1 Library Generation Phase

Random mutagenesis, optionally coupled with DNA shuffling, can leveragea full range of combinatorial amino acid replacements in multiplepositions simultaneously.

Thus, error prone PCR is used to generate a library (first levellibrary) of genes where 8 amino acid residues are randomly mutated aftertranslation per each 1 Kb of DNA (fairly homogeneous frequency ofmutation). Optionally, DNA shuffling (StEP, Staggered Extention PCR) maysubsequently be used to generate a second library (second level) withgenes randomly mutated in about 3 to 15 different amino acids per gene.Roughly 1 ng of this library will contain 1 billion genes; given theamplification method used, about 0.15 billion genes are contained ineach ng of DNA used for transformation. Depending on transformationefficiency and effort, about 1 million different genes could be screenedeach day.

EXAMPLE 2 Construct Design

The mutated genes are inserted in a pre-formed construct (plasmid form)containing:

a signal sequence,

maltose binding protein (MBP) or beta-lactamase on the receptor'sN-terminal side, and

a kanamycin or gentamicin resistance gene on its C-terminal side.

These domains are all separated by various small oligopeptide linkers.Two exemplary constructs prepared as described herein are illustrated inFIG. 2.

The construct is permanently transcribed in E. coli using constitutivepromoters (e.g. Plac) or inducible promoters (e.g. araBAD, T7) with theaddition of chemical inducers (e.g. arabinose, IPTG).

EXAMPLE 3 Selection Phase

The construct library containing modified genes is then used totransform E. coli strains (e.g. BL21 or DH10beta). Growth is tested onLB medium with varying concentrations of kanamycin (MIC foruntransformed cells is approximately 10 mg/L), both in liquid and agarplates.

An N-terminal truncation (aa 43-424) of the wild type Neurotensinreceptor 1 from rat (NTSR1, UniProt P20789) is used as a control system.

About 25 constructs were generated with wild type NTSR1 consisting ofcombinations of:

3 different signal sequences (gIIIss, DsbAss, MBPss),

2 fusion partners (TrxA, MBP),

several oligopeptide linkers, and

antibiotic resistance enzymes (NPTII for kanamycin resistance, AAC(3)-1for gentamicin resistance, TEM-1 β-lactamase for carbenicillinresistance).

Apparent MIC for both antibiotics tested (carbenicillin and gentamicin)increased considerably when E. coli expressed certain constructscontaining NTSR1 (>50 mg/L kanamycin, >75 mg/L carbenicillin).

MIC for both antibiotics tested (carbenicillin and kanamycin) alsoincreased when E. coli contained a plasmid encoding the wild typemembrane-spanning protein receptor GPR55 from human (UniProt Q9Y2T6)(approximately 25 mg/L kanamycin, 40 mg/L carbenicillin).

EXAMPLE 4 Results

Libraries of mutated GPR55 genes were created containing between about 3and 15 random mutations (residue) per gene. Selection of plasmidscontaining enhanced receptor mutants was performed at 50 mg/L kanamycin.

Transformation of E. coli strains with these libraries resulted in theisolation of mutated GPR55 clones that could confer resistance to highconcentrations of kanamycin (>50 mg/L) or carbenicillin (>80 mg/L).

Mutated clones are transferred to mammalian expression hosts, includingHEK293T by transfection using the pcDNA3.1 vector. Expression isobserved to increase by >5-fold over constructs containing wild typeGPR55, as judged by electrophoretic (see, for example, FIG. 3) and fSECtechniques.

Mutated clones are expressed and purified; the resultingprotein-detergent complex samples demonstrate an increase inthermostability of up to 7° C. over wild type after one round ofmutagenesis (as judged by fSEC (see, for example, FIG. 4) andfluorimetric techniques).

Mutated clones also demonstrate a more homogeneous oligomeric statecompared to wild type when receptor samples are purified in ligand freeform (see, for example, FIG. 5).

When using hyperthermophilic species (e.g. Thermus thermophilus) as aselection host, the protein melting temperature (in the thermalunfolding format) of the identified mutants is expected to be superiorwhen compared to a selection process performed in E. coli. (i.e., morethan 10° C. higher than wild type).

EXAMPLE 5

Antibody selection is expected to greatly improve (become easier) whenusing said thermostabilized mutants as selecting antigen in phage oryeast display (or similar antibody screening, as well as antibodygeneration). Generating antibody should also be facilitated when thesethermostabilized mutants are used as immunogen in-vivo in protein form,or using sequence encoding DNA or RNA in an appropriate animal or humanhost, or in association with an appropriate immunization vehicle orappropriate immune formulation used as an adjuvant or delivery vehicle(see, for example, Hutchings C J et al. 2014 Mabs, 6(1):246-61).

The invention illustratively described herein may be practiced in theabsence of any element or elements, limitation or limitations which isnot specifically disclosed herein. The terms and expressions which havebeen employed are used as terms of description and not of limitation,and there is no intention that in the use of such terms and expressionsof excluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method of selecting an antibody that binds to amembrane-spanning protein with one or more modulated functionalproperties, said method comprising: contacting the antibody with themembrane-spanning protein; and detecting a binding between the antibodyand the membrane-spanning protein; wherein the membrane-spanning proteinis generated by a method comprising: generating, by an in vitro method,a first polynucleotide library from a starting polynucleotide thatencodes the membrane-spanning protein, wherein a sufficient number ofbases of said starting polynucleotide is randomly modified so as tomodulate the functional properties of the membrane-spanning protein,optionally generating, by an in vitro method, a second polynucleotidelibrary from said first polynucleotide library, said secondpolynucleotide library comprising a polynucleotide encoding saidmembrane-spanning protein modified by DNA shuffling so as to modulatethe functional properties of the membrane-spanning protein, insertingthe modified polynucleotides of said first or second polynucleotidelibraries into a construct comprising: a signal sequence, a first markersequence, wherein said first marker sequence is in-frame with saidmodified polynucleotide, is downstream of said modified polynucleotide,and encodes a polypeptide that overcomes the sensitivity of a host cellto the presence of selective pressure agent(s)/conditions and thepolypeptide is fused to said membrane-spanning protein, and a secondmarker sequence, wherein said second marker sequence-is in-frame anddownstream of said signal sequence, but upstream of said modifiedpolynucleotide, is different from the first marker and encodes apolypeptide that overcomes the sensitivity of the host cell to thepresence of selective pressure agent(s)/conditions, is located in theopposite side of the membrane than the first marker, and is fused tosaid membrane-spanning protein, thereby producing modifiedpolynucleotide-containing constructs, transforming suitable host cellswith the modified polynucleotide-containing constructs; culturing thetransformed host cells in the presence of said first and secondselective pressure agent(s)/conditions, wherein theconcentration/condition level of said first and second selectivepressure agent(s)/conditions are higher than the concentration/conditionlevel of said first and second selective pressure agent(s)/conditionsused for culturing equivalent host cells transformed with the startingpolynucleotide; selecting those cells which, in contrast to the startingpolynucleotide, survive exposure to said first and second selectivepressure agent(s)/conditions; and identifying the modifiedpolynucleotide-containing construct(s) contained therein; wherein thesurviving cells contain a membrane-spanning protein having one or moremodulated functional properties relative to the starting polynucleotideencoding the membrane-spanning protein.